Planta (2006) 224: 1004–1014 DOI 10.1007/s00425-006-0283-3

ORIGINAL ARTICLE

Heping Yang · Navpreet Kaur · Stephanie Kiriakopolos Sheila McCormick

EST generation and analyses towards identifying female gametophyte-specific genes in Zea mays L.

Received: 23 January 2006 / Accepted: 2 April 2006 / Published online: 23 May 2006© Springer-Verlag 2006

Abstract The embryo sac (female gametophyte) playsan important role in double fertilization. The femalegametophyte is composed of four speciWc cell types: thesynergids that attract pollen tubes, the egg cell and cen-tral cell which are fusion partners for the two sperm cells,and the antipodal cells whose function is unknown. As aresource for gene discovery and to help identify genesexhibiting cell-speciWc expression patterns, we con-structed cDNA libraries from female gametophytes andfrom egg cells of maize and sequenced more than 8,500ESTs. These libraries represent diverse transcripts,potentially corresponding to 3,850 genes (contigs andsingletons) from the female gametophyte and 963 genes(contigs and singletons) from the egg cell. In each collec-tion, 16% of the contigs/singletons have no matches indatabases and 3–5% encode hypothetical proteins; novelhypothetical proteins (not found within the femalegametophyte contigs) were identiWed among the egg cellcontigs. We examined 65 contigs by RT-PCR and 19genes that were potentially female gametophyte-speciWcwere identiWed. We used in situ hybridization to deter-mine expression speciWcity for seven genes: one tran-script was expressed both in the egg cell and in thecentral cell, one was expressed in the egg cell and synerg-ids, two were expressed in the central cell, two wereexpressed in the synergids, and one was expressed in thecentral cell and the synergids. Four of these encode

small, potentially secreted peptides that are dissimilarexcept for a conserved triple cysteine motif near their C-terminus. These EST resources should prove useful foridentifying female gametophyte or cell-speciWc genes.

Keywords Central cell · Double fertilization · Egg cell · Gamete · Synergid · Triple cysteine motif protein

Abbreviations Cdk: Cyclin-dependent kinase · DIG: Digoxigenin · EA: Egg apparatus · EAL: EA1-like · EBE: Embryo sac/basal endosperm · eIF-5A: Eukarytic translation initiation factor 5A · ES: Embryo sac · ESR1g1: Embryo surrounding region 1g1 · EST: Expressed sequence tag · GPI-Aps: Glycosylphos-phatidyl inositol-anchored proteins · NP1: Nucellain precursor · TLA1: Transparent leaf area1

Introduction

The life cycle of higher plants alternates between thespore-producing sporophyte, a pre-dominant diploidgeneration, and gamete-producing gametophytes, areduced haploid generation. The pollen grain (malegametophyte) is composed of a large vegetative cell andtwo sperm cells. The female gametophyte typically con-sists of seven cells: the egg cell, two synergid cells, a binu-cleate central cell and three antipodal cells. Inangiosperms, double fertilization occurs. The pollen tubeenters a synergid to deliver the two sperm cells to thefemale gametophyte. One sperm cell fuses with the eggcell to form the zygote that develops into the embryo,whereas the second sperm cell fuses with the central cellto yield the primary endosperm cell that eventuallydevelops into the endosperm. Although double fertiliza-tion was discovered over 100 years ago, molecules thatcontrol this unique feature of angiosperms are largelyunexplored because of the inaccessibility of plantgametes, which are embedded in sporophytic tissues(reviewed in Weterings and Russell 2004).

Electronic Supplementary Material Supplementary material isavailable for this article at http://dx.doi.org/10.1007/s00425-006-0283-3 and is accessible for authorized users.

H. Yang · N. Kaur · S. Kiriakopolos · S. McCormick (&)Plant Gene Expression Center, United States Department of Agriculture, Agricultural Research Service, and Department of Plant and Microbial Biology, University of California at Berkeley, 800 Buchanan Street, Albany, CA 94710, USAe-mail: sheilamc@nature.berkeley.edu

Present address: H. YangMonsanto Company, 800 N. Lindbergh Blvd., St. Louis, MO 63167, USA

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Successful fertilization in angiosperms depends oncommunication between the male gametophyte andfemale Xoral organs. Sperm cells are within the vegeta-tive cell of the pollen grain and, unlike animal spermcells, are not motile but are transported to the femalegametophyte by the pollen tube. The pollen tuberesponds to multiple signals on its journey to the femalegametophyte (reviewed in Swanson et al. 2004) Forexample, genetic evidence indicated the existence of afemale gametophyte-derived, long-range activity control-ling pollen tube attraction (Hülskamp et al. 1995; Rayet al. 1997). Analyses of the Arabidopsis mutants maga-tama (Shimizu and Okada 2000), gfa2 (Christensen et al.2002), feronia (Huck et al. 2003) and sirene (Rotmanet al. 2003) suggested that signals from the female game-tophyte control pollen tube attraction, penetration,reception and subsequent sperm cell delivery. Disruptionof these signals resulted in sterility although pollen tubescould still arrive at the female gametophyte (Shimizu andOkada 2000; Huck et al. 2003; Rotman et al. 2003). Fur-thermore, cell ablation experiments demonstrated thatthe synergids are the source of attraction signal(s) whosemolecular nature is still unknown (Higashiyama et al.2001). In maize, a small protein present in the synergidsand egg cell, ZmEA1, is thought to play a role in pollentube attraction (Márton et al. 2005).

Large-scale single-pass sequencing of randomlyselected cDNA clones is useful for discovering novelgene functions, identifying novel gene family members,and deWning gene expression proWles. Comparison ofESTs from diVerent tissues within an organism helps toidentify tissue-speciWc genes. Some EST resources existfor the female gametophyte. Young ear cDNA librarieswere sequenced by the maize EST project (http://www.mutransposon.org/project/RescueMu/zmdb/est/library.php), but mRNAs from the female gametophyteare likely not well-represented in such libraries, becausemost of a young ear is composed of sporophytic tissues.A cDNA library was constructed from isolated egg cellsof maize (Dresselhaus et al. 1994). Transcripts from thatlibrary include those encoding calreticulin (Dresselhauset al. 1996), ribosomal proteins (Dresselhaus et al.1999a), eukaryotic translation initiation factor 5A (eIF-5A) (Dresselhaus et al. 1999b), defensin-like proteins(Cordts et al. 2001), egg apparatus 1 (EA1) (Márton et al.2005), and TRANSPARENT LEAF AREA1 (TLA1;Dresselhaus et al. 2005). Recently, the same groupreported (Sprunck et al. 2005) 735 expressed sequencetags (ESTs) from a wheat egg cell cDNA library, repre-senting 404 genes, most of which were metabolismrelated. Okamoto et al. (2004) identiWed eight abundantproteins in the maize egg cell, and Okamoto et al. (2005)identiWed six ESTs that were expressed in the egg cell. Leet al. (2005) prepared cDNA libraries from maize eggcells and central cells and performed a suppression-sub-tractive hybridization, followed by microarray screeningof »»1,000 cDNAs, to identify clones with enhancedexpression in one of these cell types. They identiWed andreport on »»30 ESTs from each library. They used in

situ hybridization for Wve of these, to determine cellularexpression patterns, and identiWed one apparently egg-speciWc EST and one apparently central cell-speciWcEST. The synergids are responsible for pollen tubeattraction but no synergid cDNA library exists. A func-tion for the antipodal cells is unknown—in many speciesthey degenerate in the mature female gametophyte, butin maize they proliferate—no antipodal cell cDNAlibrary exists. Yu et al. (2005) recently identiWed femalegametophyte transcripts in Arabidopsis by comparativetranscriptomics between ovules containing femalegametophytes and ovules lacking them, due to a muta-tion at the sporocyteless/nozzle gene.

We isolated female gametophytes from maize andconstructed a cDNA library, so that genes expressed inany cell type of the maize female gametophyte would berepresented. Single-pass sequencing of 7165 ESTS fol-lowed by bioinformatic analyses indicated that thisfemale gametophyte library is useful for gene discovery;the contigs represent 3,850 genes and 183 correspond togenes annotated as hypothetical proteins. To enrich foregg cell-speciWc transcripts and to determine the egg celltranscriptome, we also constructed a cDNA library fromisolated egg cells. Analysis of 1,415 egg cell ESTsrevealed that egg cells have diverse transcripts. Notably,many ESTs from the egg cell library were not identiWedwithin the female gametophyte ESTs, demonstratingthat enrichment for a single cell type is eVective at identi-fying novel ESTs. We performed RT-PCR on 65 selectedESTs from the female gametophyte library and identiWed19 putatively female gametophyte-speciWc genes. Five ofthese encode a novel family of small cysteine-rich pro-teins with a triple cysteine motif at or near the C-termi-nus; these have diVerent cellular expression patternswithin the embryo sac. By signiWcantly increasing publi-cally available ESTs from the female gametophyte andegg cell, we have provided the research community witha resource for further studies on gametophyte develop-ment and double fertilization.

Materials and methods

Isolation of female gametophytes and egg cells

Zea mays (inbred line A188) plants were grown in agreenhouse for a 16 h day, 25/19°C (day/night) and 70%relative humidity. An irradiance of 50–120 W m¡2 wasprovided by 1,000 W lamps. The Wrst ears appearing on8-week-old plants were covered with paper bags beforesilk emergence and female gametophytes were isolatedfrom these unpollinated ears that had been covered for2 weeks.

Female gametophytes were isolated by enzymaticmaceration of ovule slices followed by manual microdis-section, essentially as described in Kranz et al. (1991).BrieXy, unfertilized ears (on average, 15 cm in lengthwith 10 cm long silks, including both visible and those

1006

covered by husks) were harvested, the husks were sur-face-sterilized with ethanol (70%), then husk leaves wereremoved and ovaries collected from the middle of theears. Ovaries were longitudinally cut, while observingunder a dissecting scope, to obtain pieces containingfemale gametophytes. The pieces were kept in 0.53 Mmannitol solution, pH 5.0 before enzymatic maceration.Approximately, 20 ovule pieces were incubated (25°C,»»1 h) in a 3.5-cm diameter plastic dish with 1.5 ml ofenzyme mixture containing 0.75% pectinase (Sigma, StLouis, MO), 0.25% pectolyase Y23 (Seishin, Tokyo,Japan), 0.5% hemicellulase (Sigma), and 0.5% cellulaseOnozuka RS (Yakult Honsha, Tokyo, Japan). Femalegametophytes were isolated with microdissection nee-dles. To isolate egg cells, the female gametophyte wallwas cut with microdissection needles. Female gameto-phytes and egg cells were collected into 1.5 ml Eppendorftubes with disposable transfer pipettes (1.5 £ 30 mm)(BIO-RAD, Richmond, CA) and rinsed with 0.53 Mmannitol solution three times. The extra mannitol solu-tion was removed after centrifugation at 3,000 rpm for1 min. Isolated female gametophytes and egg cells werefrozen in liquid nitrogen and stored at ¡80°C.

RNA extraction and library construction

Total RNA was extracted from 300 embryo sacs and 270egg cells using 0.5 ml TRIzol Reagent (GIBCO BRL,Gaithersburg, MD) and quantiWed with Ribogreen(Molecular Probes, Eugene, OR). Total RNA was usedto synthesize cDNA; cDNA libraries of female gameto-phytes or egg cells were constructed by oligo dT primingusing a SMART PCR cDNA synthesis kit (Clontech,Palo Alto, CA). The resulting PCR-ampliWed cDNA wasdigested with SWI and ligated to SWI-cut lambda arms;the cDNA was oriented in the vector. The recombinantswere packaged with Gigapack III Gold packagingreagents (Stratagene, La Jolla, CA). The primary cDNAlibraries were used as the EST source.

Plasmid isolation and sequencing

Individual phage clones were converted into plasmids inEscherichia coli (strain BM25.8) using the Clontech CreloxP system. Plasmid DNA was extracted from BM25.8cells using DirectPrep 96 MiniPrep Kits (QIAGEN,Valencia, CA). cDNA inserts of 300 bp or longer wereampliWed from the 5� end using the Clontech 5� endsequencing primer that Xanks the cDNA inserts and aBigDye Terminator Cycle Sequencing Kit (Applied Bio-systems, Foster, CA). PCR products were sequencedwith an ABI PRISM™ 3100 Genetic Analyzer (AppliedBiosystems). If there were more than three Ns in a row,the sequence thereafter was removed to control sequencequality. The female gametophyte EST sequences are inGenBank; accession numbers are listed in Table S1.Sequencing is ongoing: updated information can beviewed at the McCormick lab web site, http://www.pgec.usda.gov/McCormick/mclab.html, or at NCBI

(http://www.ncbi.nlm.nih.gov) using the query “Zeamays embryo sac” and “Zea mays egg cell”.

Bioinformatics

Ambiguous, linker, and vector sequences were removedmanually prior to bioinformatic analysis. Sequences wereassembled with Sequencer™ 3.1.2 using the default set-tings. For annotation, manually processed sequences werecompared against current public databases, Wrst usingBlastx (http://www.ncbi.nlm.nih.gov). If a sequence was tooshort to obtain matches in the protein databases, we usednucleotide-nucleotide blast (Blastn) to attempt to obtain alonger EST sequence and then re-ran Blastx with thelonger query. All parameters used default values. The ESTswere manually annotated using the information returnedby Blastx. A transcript was assigned a match to a proteinof known identity if the probability of similarity score inBlastx was at the threshold e value of 10¡8 or smaller. Thematch with the lowest score was chosen when an ESTsequence matched more than one gene, as in the case ofgene families. Amino acid sequences of the triple cysteinemotif family were compared and an alignment was gener-ated using Multalin (Corpet 1988). Contig assembly wascarried out with Sequencer™ 3.1.2 using the default set-tings. Contigs were assigned functional categories based onGene Ontology (Camon et al. 2003), after sequence com-parisons of their predicted gene products with the currentSWISS-PROT/TrEMBL (SWALL) databases.

Reverse transcription (RT)-PCR analysis

To test the purity of the female gametophyte RNA, gene-speciWc primers (Table S3) were used to amplify tran-scripts of embryo sac (ES) 1 and ES2/3/4 (positive con-trols), Nucellain precursor¡1 (NP1) (Linnestad et al.1998) and embryo surrounding region 1g1 (ESR1g1)(negative controls). The NP1 primers ampliWed the pre-dicted size product from maize ovule slices (not shown).For analysis of multiple tissue expression patterns, totalRNA was isolated from ovule slices with female gameto-phytes, ovule slices with nucellar cells only, silks, leaves,roots and pollen of 8-week-old maize plants. The RNAwas treated with DNase I and then individually used tosynthesize Wrst strand cDNA using the Moloney MurineLeukemia Virus (MMLV) reverse transcriptase suppliedin the First-strand cDNA synthesis kit (Invitrogen,Carlsbad, CA). First strand cDNA (50 ng) from each tis-sue was ampliWed with transcript-speciWc primers. PCRwas run for 30 cycles; denaturation at 95°C for 30 s,primer annealing at 55°C for 30 s, and polymerization at72°C for 3 min, in a Thermal Controller (MJ Research,Watertown, MA). RT-PCR products were separated byelectrophoresis on a 1% agarose gel in 0.5£ BE buVer.

In situ hybridization

Female gametophyte-speciWc cDNA inserts were clonedinto pGEM 3Zf vector (Promega, Madison WI). Linear-

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ized DNA templates (2 �g) were used to synthesize senseand antisense probes with SP6 or T7 RNA polymerasefrom the Digoxigenin (DIG) RNA labeling kit (RocheApplied Science, Indianapolis, IN). RNA probes longerthan 500 bp were hydrolyzed to »»500 bp fragments infresh 0.1 M carbonate buVer (pH 10.2). DIG-labeledRNA probes were resuspended in 50% formamide andstored at ¡20°C.

Ovule pieces containing female gametophytes wereWxed in 4% paraformaldehyde in 1£ PBS at 4°C for 36 h,and then dehydrated at 4°C through a graded ethanol:H2O series. The tissues were then placed in 75% EtOHand 25% Histoclear (National Diagnostics, Atlanta, GA)for 1.5 h, 50% EtOH and 50% Histoclear for 1.5 h, 25%EtOH and 75% Histoclear for 1.5 h, and 100% Histoclearfor 3 £ 1.5 h, all at room temperature. After Histoclearwas gradually replaced with molten paraplast at 50°C,tissues were transferred into molds and stored at 4°C orused directly. ParaYn-embedded tissues were sectionedon a microtome to 9 �m thickness. Pretreatment, hybrid-ization and washing (at 50°C), and detection were per-formed as described (Torres et al. 1995), except that theantibody (anti-digoxigenin-alkaline phosphatase fabfragments) was purchased from Roche Applied Science.Images were captured from a Zeiss Axiophot microscope(Thornwood, NY) with a SPOT camera (DiagnosticInstruments, Sterling Heights, MI).

Results

Quality of female gametophyte and egg cell cDNA libraries

We collected unfertilized maize ears and isolated 300female gametophytes (Fig. S1). Dynamic cytoplasmicstreaming in all the cells was visible, indicating that theisolated female gametophytes were viable. Each isolatedfemale gametophyte still had a few nucellar cellsattached; nucellar cells are from the sporophyte andRNA from them was therefore not wanted. Because itwas too tedious to manually remove these attachednucellar cells, we tested the purity of the RNA from theisolated female gametophytes to determine if RNA fromthe few remaining nucellar cells could be detected. Weperformed RT-PCR with primers speciWc for the femalegametophyte-expressed genes ES1 and ES2/3/4 (Cordtset al. 2001), a nucellar cell-speciWc gene, NP1 (Linnestadet al. 1998) and an endosperm-speciWc gene ESR1g1(Opsahl-Ferstad et al. 1997). Figure 1 shows that onlytranscripts from the ES1 and ES2/3/4 genes weredetected. Although we cannot completely exclude thepresence of transcripts from nucellar-expressed genes, weconcluded that the female gametophyte RNA was pureenough for cDNA library construction. We then synthe-sized cDNA using total RNA from female gametophytesand constructed a library using a PCR-based method.From one ligation and packaging we obtained about

225,000 pfu. To assess the quality of this library, wetested 116 randomly selected plaques: about 90% hadcDNA inserts; insert sizes ranged from 0.3 to 2.9 kb, withthe majority falling between 0.5 and 0.9 kb.

Egg cells were isolated mechanically from isolatedfemale gametophytes without using additional cell wall-degrading enzymes, to minimize stress responses(Fig. S2). We collected 270 egg cells and constructed acDNA library from total RNA. From one ligation andpackaging we obtained 325,000 pfu. We randomlyselected 192 clones to assess the quality of this library:about 95% had cDNA inserts; insert sizes ranged from 0.3to 3.2 kb, with the majority falling between 0.5 and 1.3 kb.

Diversity of transcripts

EST clones from the unampliWed libraries were ran-domly picked and those with inserts larger than 300 bpwere sequenced from their 5� ends in a single run. ESTsequencing from these libraries is ongoing; for updatedinformation go to http://www.pgec.usda.gov/McCormick/mclab.html, or search Genbank (http://www.ncbi.nlm.nih.gov) with the query “Zea mays embryo sac” or “Zeamays egg cell”.

About 30% of 7,165 female gametophyte ESTs werefull length, or judged to be so based on size of insert and/or alignment with the closest match in the databases. Amajority of the EST clones encoded full open readingframes (ORFs) or C-terminal coding regions with 3�UTRs; less than 10% were 3� UTRs of known or pre-dicted genes. In order to assess the level of redundancyand to estimate the number of unique genes potentiallyrepresented in the ESTs, we assembled them into contigs.

Fig. 1 Purity of isolated female gametophytes. Total RNA fromisolated female gametophytes was used to synthesize cDNA. RT-PCR was performed with primers speciWc for ES1(1), ES2/3/4 (2),NP1 (3), and ERS1g1(4). The expected sizes are indicated with aster-isks. Molecular weight marker is �/HindIII (M)

M 2 3 41

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Table S1 lists the contigs assembled from 7,165 ESTsand includes accession numbers, functional category andbest BLASTP match. About 40% of the ESTs were sin-gletons. The rest assembled into contigs of 2–188 clones.Figure 2 shows the 20 largest contigs from the femalegametophyte. Each contig is composed of more than 20overlapping or redundant ESTs. The majority of theseclusters correspond to housekeeping genes. Overall, thecurrent contigs and singletons correspond to potentially3850 unique genes, indicating that the female gameto-phyte has diverse transcript populations.

The contigs were grouped according to functionalcategories as summarized in Fig. 3; over 70% encodeknown proteins and their functional categories are typi-cal of the representations seen in other cDNA librariesor transcriptomes from various tissues (Hennig et al.2004; Sprunck et al. 2005). Metabolism-related genes arethe most frequently represented while genes involved incell growth/division were least abundant. About 12% ofthe contigs were classiWed as “unknown protein” or“expressed protein” by comparison to sequences fromrice, Arabidopsis or other species. The best matches for4.7% of the contigs are to genes that are annotated asencoding hypothetical proteins in the Arabidopsis, rice orother genomes. Those with signiWcant matches to Ara-bidopsis hypothetical proteins are indicated in Table S1;some others were similar to hypothetical proteins fromrice, including 13 cysteine-rich proteins analyzed furtherbelow. Of the 989 ESTs with no signiWcant matches, 386assembled into 107 contigs while the others were single-tons. Most of these contain an ORF; some of the restmight correspond to noncoding RNAs. When combined,these three categories (encoding unknown, hypotheticalor novel proteins) constitute 30% of the contigs.

Diverse transcripts were also represented in the eggcell cDNA library (Table S2). Of the 1,415 ESTs, 806were singletons and the other 609 ESTs assembled into156 contigs, thus potentially representing 962 genes.Many of the most abundant contigs show no signiWcantsimilarity to the databases, or correspond to unknownproteins (Fig. 2b). Overall, the functional distributions ofthe egg cell ESTs were similar to those of the femalegametophyte (Fig. 3). Within the no signiWcant similar-ity, hypothetical protein and unknown protein groups,there was limited overlap between the female gameto-phyte and egg contigs.

IdentiWcation of genes potentially speciWc to the female gametophyte

We reasoned that cDNAs that were female gametophytespeciWc might be absent or underrepresented in currentEST databases. Because there are extensive ESTs fromlibraries made from diverse tissues of maize (http://www.tigr.org/tigr-scripts/tgi/libtc.pl?db=maizest) andmost of our ESTs fell into a contig with these alreadysequenced ESTs, we did not randomly select ESTs andperform PCR to determine their expression patterns.

Fig. 2 Summary of the 20 largest contigs (> 20 ESTs) assembledfrom female gametophyte ESTs (a) or egg cell ESTs (b). The Gen-bank accession number listed after each name is of the best match inthe database. Polyubiquitin 4 = S28426; Glycosyltransferase =XP_475798.1; Ribosomal protein L35A (60S) = AAL59231.1; GPI-anchored protein = CAE05527.1; Golgi-associated protein se-wap41 = T04331; Asparate aminotransferase = NP_916216.1;No signiWcant similarity = ES12381 (DN828382); Unknownprotein = XP_468711.1; Calmodulin = CAA46150.1; Alcoholdehydrogenase = AAA33434.1; Hypoxia-responsive protein =NP_198128.1; RAFTIN1 protein = XP_465009.1; CytochromeP450 = NP_922325.1; Translation factor SUI1 = P56330; Hypo-thetical protein = Q8LHP0; Beta-expansin (EXPB15) =AAM73779.1; Thioredoxin, H-type = AAL67139.1; Water stress-induced protein = T07613; Unknown protein = AAF98579.1; Ear-ly drought-induced protein = AAM46894.1; ES1 = AAK08132.1;ES3 = AAK08134.1; No signiWcant similarity C (triple cysteine mo-tif containing) = E1872 (DT535575); AE1 = CAB56552.1; Un-known protein F = XP_479340.1; Unknown protein E =XP_479340.1; Chitinase = T03405; Germin A = XP_480464.1;Glutathione peroxidase = NP_192897.2; Thioredoxin H-type = T04090; Unknown protein D = CAE04145.1; Unknownprotein C = NP_568713.1; Unknown protein B = XP_473116.1;Unknown protein A = NP_568088.1; No signiWcant similarityB = E0002(DR451926); No signiWcant similarity A = E0017(DR451941); Germin-like = XP_480451.1; Subtilisin/chymotrypsininhibitor = S61830; Glutathione transferase = NP_909709.1;Bowman–Birk serine protease inhibitor = NP_910046.1

Number of ESTs

Early drought induced proteinUnknown protein

Water stress-induced proteinThioredoxin H-type

Beta-expansin (EXPB15)Hypothetical protein

Translation factor SUI1Cytochrome P450RAFTIN1 protein

Hypoxia-responsive proteinAlcohol dehydrogenase

Unknown proteinCalmodulin

No significant similarityAsparate aminotransferase

Golgi-associated protein se-wap41GPI-anchored protein

Ribosomal protein L35A (60S)Glycosyltransferase

Polyubiquitin 4

0 50 100 150 200

Bowman-Birk serine protease inhibitorGlutathione transferase

Subtilisin/chymotrypsin inhibitorGermin-like

No significant similarity ANo significant similarity B

Unknown protein AUnknown protein BUnknown protein CUnknown protein D

Glutathione peroxidaseThioredoxin H-type

Germin AChitinase

Unknown protein EUnknown protein F

AE1No significant similarity C

ES1

a

b ES3

Number of ESTs

ES

T c

on

tig

ES

T c

on

tig

Early drought induced proteinUnknown protein

Water stress-induced proteinThioredoxin H-type

Beta-expansin (EXPB15)Hypothetical protein

Translation factor SUI1Cytochrome P450RAFTIN1 protein

Hypoxia-responsive proteinAlcohol dehydrogenase

Unknown proteinCalmodulin

No significant similarityAsparate aminotransferase

Golgi-associated protein se-wap41GPI-anchored protein

Ribosomal protein L35A (60S)Glycosyltransferase

Polyubiquitin 4

0 50 100 150 200

Bowman-Birk serine protease inhibitorGlutathione transferase

Subtilisin/chymotrypsin inhibitorGermin-like

No significant similarity ANo significant similarity B

Unknown protein AUnknown protein BUnknown protein CUnknown protein D

Glutathione peroxidaseThioredoxin H-type

Germin AChitinase

Unknown protein EUnknown protein F

AE1No significant similarity C

ES1

Number of ESTs0 4020 60 80

b ES3

1009

Indeed, when we checked expression patterns for ES0916(eIF-5A) and ES0904 (blue copper-binding protein), wefound that our RT-PCR results closely paralleled ESTdatabase representation. Instead we selected candidatesfor RT-PCR from those ESTs that had no databasematches, or that encoded hypothetical proteins, or thatencoded unknown proteins whose EST matches in publicEST databases were only from reproductive tissues (Xow-ers, ovaries, ovules, embryos and endosperm). Usingprimers speciWc for the ES1 and ES2/3/4 genes, we WrstconWrmed that these female gametophyte-speciWc mes-sages (Cordts et al. 2001) could be detected in cDNAsderived from ovule slices with female gametophytes, andwere not detected in cDNAs derived from ovule sliceslacking female gametophytes (data not shown).

Of 65 genes tested by RT-PCR, 19 appeared to befemale gametophyte speciWc (Fig. 4), although genesexpressed in nucellar cells surrounding the femalegametophytes might also be represented in this group.Ten of these ESTs (ES0465, ES1086, ES2711, ES3441,ES3465, ES3793, ES5686, ES8328, ES8389, andES10996) did not have any match in the current data-bases, Wve (ES1411, ES5963, ES6088, ES6468 andES7809) encode hypothetical proteins, and two (ES0965and ES2488) encode unknown proteins. The last two(ES0777 and ES0168) were similar to previouslydescribed proteins. ES0777 shares similarity with themaize ae1 protein. The ae1 gene was expressed at a veryearly stage of androgenic embryogenesis in vitro and inearly endosperm in vivo (Magnard et al. 2000); ourresults show that a similar protein is also expressed in theunfertilized female gametophyte. ES0168 encodes a pro-tein similar to embryo sac/basal endosperm (EBE)-2(CAD24798.1). ZmEBE-2 is expressed in the femalegametophyte and in the basal endosperm transfer layerof maize (Magnard et al. 2003).

Nine of the 19 putatively female gametophyte-speciWcESTs (ES0777, ES1411, ES2711, ES3441, ES3465,

ES3793, ES5686, ES5963 and ES10996) encode smallcysteine-rich proteins. Five of these (ES2711, ES3441,ES3465, ES5686 and ES10996) display very low identitywith each other, but their cysteine residues, including atriple cysteine motif at or near their C-termini (Fig. 5),are conserved among these proteins. All of these triplecysteine motif-containing proteins have a predicted N-terminal signal peptide, suggesting that they are secreted.The deduced amino acid sequences of ES1411, ES3793and ES5963 are also cysteine-rich; however, they do nothave the triple cysteine motif and do not share any simi-larity to each other. The deduced amino acid sequencesof the other 10 putatively female gametophyte-speciWcESTs are not cysteine-rich: ES0465 and ES1086 containsmall ORFs while ES8328 and ES8389 have long ORFs.ES0965 encodes a protein similar to an Arabidopsisunknown protein (At1g21065). ES2488 is similar to anunknown protein (NP_913003.1) in rice. ES6088 is simi-lar to a hypothetical protein (susceptibility homeodo-main transcription factor) conserved in rice andArabidopsis (At1g56580). ES6468 and ES7809 are similarto hypothetical proteins found only in rice.

Other tested clones showed diVerent expression pat-terns (Fig. 4). Four ESTS (ES0368, ES0411, ES3735 and

Fig. 3 Functional classiWcation of maize egg cell (open bars) and fe-male gametophyte (Wlled bars) contigs. The contigs listed inTables S1 and S2 were grouped into diVerent categories based ontheir biological functions

0.00 5.00 10.00 15.00 20.00

Cell Structure

Defense/Stress Responses

Energy

Hypothetical Proteins

Intracellular Traffic

Metabolism

No significant similarity

Protein Destination & Storage

Protein Synthesis

Signal Transduction

Transcription & Post-Transcription

Transporters

Transposons

Unclassified Function

Unknown Proteins

Percentage 0.00 5.00 10.00 15.00 20.00

Cell Growth/Division

Cell Structure

Defense/Stress Responses

Energy

Hypothetical Proteins

Intracellular Traffic

Metabolism

No significant similarity

Protein Destination & Storage

Protein Synthesis

Signal Transduction

Transcription & Post-Transcription

Transporters

Transposons

Unclassified Function

Unknown Proteins

Percentage

Fig. 4 Tissue-speciWc expression of selected genes. Total RNA fromovule slices with female gametophytes (ES), ovule slices lacking fe-male gametophytes (N), silks (S), Anther (A), leaves (L) and roots(R) was used to synthesize cDNA. PCR was carried out and theproducts were separated by electrophoresis. For genes expressed inseveral tissues, only one representative from each expression patternis shown. a A diagram showing how the cuts were made. b RT-PCRproducts

FG

N

ES0136

ES0411

ES0040

ES1244

ES3024

ES0368

ES0904

ES2773

ES3735

ES8509

ES5119

ES0913

ES8975

ES8775

ES7846

ES3683

FG N S P L R

FG N S P L R

ES6468

ES8389

ES10996

ES8328

ES7809

ES6088

FG

N

FG

N

b

ES0965

ES0465

ES0168

ES2711

ES3441

ES3465

ES0777

ES5686

ES1411

ES5963

ES3793

ES1086

ES2488

a

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Fig. 5 Alignment of amino acid sequences of the triple cysteine mo-tif family. The amino acid sequences deduced from ES2711, ES3441,ES3465 and ES5686 were used to search TIGR (http://www.tigr-blast.tigr.org/tgi/) EST databases; retrieved EST sequences that con-tained a region encoding the triple cysteine motif were translatedand aligned using Multalin (Corpet 1988). Single-letter abbrevia-tions for the amino acid residues are used. Gaps are shown as dashes.

Clone numbers from Arabidopsis, maize, rice and wheat are in bolditalic, bold, italic, and underlined italic, respectively. Amino acidsthat are conserved in a majority of the sequences across species areindicated in the consensus. Clone numbers from the female gameto-phyte and the egg cell libraries are in underlined bold; those in nor-mal typeface are from barley

1011

ES5119) were expressed in ovule slices containing femalegametophytes and in ovule slices lacking female gameto-phytes—presumably these genes are expressed in thenucellar cells. ES0368 encodes a protein similar to anArabidopsis hypothetical protein (At5g66550). ES0411shares similarity with a rice hypothetical protein(CAE05527.1) and with an Arabidopsis GPI-anchoredprotein (At5g63500). ES3735 and ES5119 could encodesmall proteins (80 and 49 amino acids, respectively) thatdo not have matches in current databases. Some genes,such as ES0040, ES0904 and ES2773, were expressed inmost tissues but not in pollen. Others were detected in alltissues tested. ES3024 was expressed at nearly identicallevels in all tissues.

Cell speciWcities of potentially female gametophyte-speciWc genes

The RT-PCR results shown in Fig. 4 did not distinguishcell speciWcity. We therefore used in situ hybridization todetermine in which cells selected genes were expressed.For each probe, we examined 96 serial slices from sixovule pieces containing a female gametophyte. Since sliceswere 9 �m thick, not every slice went through a femalereproductive cell, i.e. only one or two slices included theegg cell or synergid cells, and only 3–6 slices included thecentral cell. Therefore, there was no signal in most of the96 serial slices. Figure 6 shows representative slices wherethere was a signal. Strikingly, diVerent members of the tri-ple cysteine family showed diVerent expression patterns.ES2711 was expressed in both the egg cell and central cell(Fig. 6a). ES3441 was expressed in the central cell(Fig. 6c). ES3465 was expressed in the egg cell and synerg-ids (Fig. 6e), and the transcripts of ES5686 were detectedin synergids (Fig. 6g). The Wfth triple cysteine gene wasnot tested. Three other tested genes also exhibited distinctexpression patterns: ES0777 transcripts were detected inthe central cell, transcripts for ES0965 were detected inthe synergids, and ES0168 was expressed in both the cen-tral cell and the synergids (data not shown).

Triple cysteine motif-containing proteins are found in many plants

Because several members of the triple cysteine familyshowed intriguing expression patterns within the embryosac, we searched databases to identify other triple cyste-ine members. TBLASTN searches were carried outagainst EST databases (http://www.tigrblast.tigr.org/tgi). No matching sequences were obtained when thefull-length amino acid sequences were used as queries.However, when we used the triple cysteine motif and 3–5 amino acids N-terminal to it and set the expectthreshold at 1,000, we identiWed a relatively large num-ber of ESTs, from a variety of species, that couldencode triple cysteine motif-containing proteins withpredicted N-terminal signal sequences. Figure 5 showsan alignment of some triple cysteine motif-containingproteins. A similar query was used to search TAIR AGI

proteins (http://www.Arabidopsis.org/Blast) and addi-tional members were found among hypothetical pro-teins. There are at least 10 members of the triple

Fig. 6 Cell-speciWc expression of triple cysteine motif genes. DIG-labeled antisense probes (a, c, e, g) and sense probes (b, d, f, h) ofES2711 (a, b), ES3441 (c, d), ES3465 (e, f), and ES5686 (g, h) wereused to detect transcripts on ovule slices. Each probe was hybridizedwith 96 serial slices from six ovule pieces containing a female game-tophyte. Bars equal 100 �m. a antipodal cells; cc central cell; e eggcell; s synergid

Antisense Sense

1012

cysteine motif family in the Arabidopsis genome(At2g20070, At2g20465, At2g24693, At2g25305, At2g42885,At3g04540, At3g16895, At5g42235, At5g54220 andAt5g55132).

Discussion

Although there are extensive ESTs from libraries madefrom diverse tissues (http://www.tigr.org/tdb/tgi/plant.shtml), transcripts speciWc to reproductive cells areunderrepresented. Recently, transcriptional proWling(Yu et al. 2005) and mutant analyses (Pagnussat et al.2005) were used to identify Arabidopsis genes that areinvolved in female gametophyte development and func-tion. Here we described sequencing and bioinformaticsanalyses of ESTs from maize female gametophyte andegg cell cDNA libraries. The maize female gametophyteand egg cell have diverse transcripts. Enzymes related tometabolism were highly represented, an observationalso made from analysis of »»750 wheat egg cell ESTs(Sprunck et al. 2005). Genes involved in cell growth/division were least abundant in the female gametophyte,not surprising because cells in the mature female game-tophyte do not divide before fertilization. ESTs encod-ing transcription and translation factors wereoverrepresented in the female gametophyte library;these results are consistent with recent expression proWl-ing of reproductive tissues and comparisons to other tis-sues (Hennig et al. 2004). Defense-related transcriptswere abundant in the female gametophyte (Table S1),egg cell (Table S2) and the wheat egg cell libraries (Spr-unck et al. 2005), although it is possible that some stress-related transcripts might have been induced during iso-lation of the female gametophytes; a similar interpreta-tion was used to explain the prevalence of a cold-induced transcript in the sperm cell library (Engel et al.2003). However, it is also possible that defense systemsare active in the female gametophyte in vivo (Heslop-Harrison et al. 1999).

A few female gametophyte-speciWc genes have beenreported in maize (Cordts et al. 2001; Magnard et al.2003; Márton et al. 2005). We obtained several ESTsidentical or similar to these previously known femalegametophyte-expressed genes, which validates ourlibrary. However, one of our objectives was to identifynew female gametophyte-expressed genes potentiallyinvolved in pollen tube attraction and gamete interac-tions. Therefore, when we selected candidates for RT-PCR, we particularly focused on small and secreted pro-teins that might be involved in cell-to-cell interactions.Because this pre-selection strategy was successful (30%of the genes tested, Fig. 4), extending the strategy toother ESTs should prove fruitful.

Some transcripts already known to be egg cell-speciWcwere enriched in the egg cell ESTs, relative to their abun-dance in the female gametophyte ESTs. For example,only 1/7165 of the female gametophyte ESTs encoded an

ES2 protein (Cordts et al. 2001). By contrast, more than1% of our egg cell ESTs encode ES proteins (Cordts et al.2001). We found no EA1 transcripts (Márton et al. 2005)in 7,165 female gametophyte ESTs but found four ESTsencoding EA1 in 1,415 egg cell ESTs. There were 7/1,415ESTs encoding one member of the triple cysteine proteingene family in the egg library, but only ES6993 (1/7,165)corresponded to this member, suggesting that it may beegg cell-speciWc. About 3% of the egg cell ESTs encodegermin-like (oxalate oxidase) proteins. By contrast, therewere only 2/7,165 female gametophyte ESTs encodinggermin-like proteins. The most abundant egg cell contig(Fig. 2b) encodes a protein similar to Bowman-Birk ser-ine protease inhibitor, while only eight of these ESTswere sequenced from the female gametophyte library. Onthe other hand, the most abundant contig from thefemale gametophyte (Fig. 2a) encodes a protein anno-tated as early drought-induced, while only four of theseESTs were sequenced from the egg cell library. Most ofthe egg cell contigs that encode hypothetical proteins orunknown proteins, or that have no matches in currentdatabases are not yet represented in the female gameto-phyte contigs. These Wndings suggest that transcriptsfrom the egg cell were underrepresented even in thefemale gametophyte cDNA library. There is no overlapbetween the best Arabidopsis matches for hypotheticalproteins in our datasets (Tables S1, S2) and the 22 hypo-thetical proteins indentifed by Yu et al (2005), as pointedout by Yu et al. (2005) in their Table S4. There is nooverlap between the genes identiWed as having defects infertilization (see Table S2 in Pagnussat et al. 2005) andthe best Arabidopsis matches to our contigs (Tables S1,S2). Similarly, the subtraction procedure used by Le et al.(2005) identiWed a few ESTs not yet sequenced from ourlibraries.

There were several instances where diVerent membersof a particular gene family were diVerentially expressedin the female gametophyte or in sperm cells (Engel et al.2003). For example, within the multi-gene family thatencodes expansins (Lee et al. 2001) the beta expansinsEXP14, EXP15, and EXP10 had no EST representation.ESTs for EXP14 and EXP15 were in the female gameto-phyte library while EXP10 was represented in the spermcell ESTs; these results suggest that EXP14/EXP15 andEXP10 are gametic cell-expressed. Another example isglycosylphosphatidyl inositol-anchored proteins (GPI-APs); GPI-APs are represented in the female gameto-phyte ESTs and in the egg cell ESTs but not in the spermcell ESTs (Engel et al. 2003). In mammals (GPI-APs) areegg cell surface proteins that are important in egg cell-sperm cell interactions (AlWeri et al. 2003).

It is expected that a given cell would have all compo-nents for a biochemical pathway or process. We surpris-ingly found that that was not always the case. Forexample, ESTs encoding seven large subunit ribosomalproteins and seven small subunit ribosomal proteinswere represented in both the female gametophyte andsperm cell ESTs. However, ESTs encoding 17 largesubunit and 9 small subunit ribosomal proteins were

1013

represented only in the female gametophyte ESTs, whilethose for one large subunit ribosomal protein (L20) andone small subunit ribosomal protein (S14) were repre-sented only in the sperm cell ESTs (Engel et al. 2003). Wecannot exclude that depth of sequencing might inXuencethe EST representation, but given that we found 28 ESTsencoding L35A in the female gametophyte ESTs, if L20and S14 were expressed in the female gametophyte, itseems reasonable that we should have found at least oneEST for L20 and S14. Perhaps the male and femalegametes bring diVerent ribosomal protein transcripts tothe zygote.

Communication between the pollen tube and thefemale gametophyte controls the growth of the pollentube towards its target, the female gametophyte. Thesignaling mechanisms are largely unknown but bothsurface-localized receptors and small molecules arelikely involved. Although there are more than 600receptor-like kinases in Arabidopsis genome, only a fewligands have been identiWed (reviewed in Torii 2004).Among the presumed female gametophyte-speciWcgenes, we identiWed a novel gene family that is predictedto encode small, secreted and cysteine-rich proteins.Given their expression patterns (Fig. 6), these proteinsare potential candidates for signaling roles, either aspollen tube attractants or for gamete interactions, as asmall cysteine-rich protein is the ligand for the receptorkinase that mediates self-incompatibility (Schopferet al. 1999), and small cysteine-rich proteins bind theextracellular domains of pollen receptor kinases (Tanget al. 2002, 2004). Eight of the triple cysteine motif-con-taining proteins in Arabidopsis (At2g24693, At2g25305,At2g42885, At3g04540, At3g16895, At5g42235,At5g54220, and At5g55132) were not predicted as indi-vidual genes in the Wrst annotation (The ArabidopsisGenome Initiative 2000). Additional members may bemissed in the current annotation.

This female gametophyte cDNA library, this egg cellcDNA library and these two EST databases are usefulresources. In addition, the female gametophyte and eggcell ESTs provide expression evidence that over 350proteins previously annotated as hypothetical in Ara-bidopsis and/or rice are in fact unknown proteins,whose functions in the female gametophyte can now beexplored. The ESTs reported here, as well as otherresources (Le et al. 2005; Pagnussat et al. 2005; Yu et al.2005) can serve as cellular markers (Fig. 6) to studyfemale gametophyte development and the promoters ofsuch genes can be used to manipulate gene expression.In the future, these ESTs can be used to develop femalereproductive cell-oriented gene chips to help identifythe genes responsible for female reproductive mutantsand to monitor changes in gene expression duringgametophyte development, double fertilization andearly embryogenesis.

Acknowledgments We thank Priti Patel, Michelle Meador, TeresaMok, Jessica Kim and Jungsun Lee for technical assistance, and allmembers of our laboratory for useful discussions. This work was

supported by the National Science Foundation (Plant Genomegrant no. 0211742).

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